Heart failure is a leading cause of death world-wide, and despite advances in drug treatment, morbidity and mortality remain high. Cardiac resynchronization (CRT) a device-based therapy in which failing hearts are bi-ventricularly stimulated to offset a conduction delay and discoordinate motion, is the first new treatment advance of the millennium, and only therapy to date to acutely and chronically enhance systolic function, while reducing long-term mortality. Despite widespread clinical use of CRT, little is known about how it works at the basic level, contributing to ongoing difficulties in its optimal deployment. We developed a dog model of dyssynchronous heart failure (DHF) and CRT that has reveal marked effects of CRT on reversing regional stress signaling and genome-wide expression heterogeneity, improving cell survival, myocyte electrophysiology, contractile function, calcium homeostasis, mitochondrial function (greatly altering its subproteome), and reversing beta-adrenergic down-regulation at multiple levels of the cascade. New data shows CRT acts as a Ca2+-sensitizer, has novel anti-oxidant effects, improves energy status, and alters mitochondrial oxidative phosphorylation linked to novel post-translational modifications (PTM). Strikingly, we now show improved myocyte function and reserve with CRT is not replicated in cells from hearts that develop failure synchronously; i.e. CRT induces specific changes when implemented in DHF. The goal of this proposal is to elucidate the mechanisms for these changes. Project 1 focuses on myofilament-Ca2+ regulation, and beta-adrenergic signaling, particularly changes in inhibitory G-protein coupling. Project 2 tests the influence of CRT on oxidant-modulated electrical instability and elucidates mechanisms for improved calcium cycling. Project 3 uses state-of-the-art proteomic analysis to explore modifications in mitochondrial ATP synthetic and redox regulatory proteins and their impact on organelle function. Using state-of-the art methodologies, including a new mouse-model of DHF and CRT, proteomic approaches to assess tiny protein amounts, and comparisons between experimental data and human myocardial tissue results, we will advance our understanding of this therapy and the patients in which it is best used.